Nitric Oxide (NO) was originally identified in vascular endothelial cells (Palmer et al. (1987) Nature 327: 524-526 and Palmer et al. (1988) Nature 333: 664-666) and has been identified as being identical to endothelium-derived relaxing factor (Moncada et al. (1989) Biochem. Pharmacol. 38: 1709-1715; Furchgott (1990) Acta Physiol. Scand. 139: 257-270 and Ignarro (1990) Annu. Rev. Pharmacol. Toxicol. 30: 535-560). Besides endothelial cells, NO formation has been demonstrated in macrophages (Hibbs et al. (1987) Science 235: 473-476 and Marletta et al. (1988) Biochemistry 27: 8706-8711), neutrophils (McCall et al. (1989) Biochem. J. 262: 293-297; Salvemini et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6328-6332 and Wright et al. (1989) Biochem. Biophys. Res. Commun. 160: 813-819), some tumor cells (Amber et al. (1988) J. Leuk. Biol. 44: 58-65), adrenal glands (Palacios et al. (1989) Biochem. Biophys. Res. Commun. 165: 802-809), Kupffer cells (Billiar et al. (1989) J. Exp. Med. 169: 1467-1472) and in brain tissue (Garthwaite et al. (1988) Nature 336: 385-388; Knowles et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5159-5162 and Bredt and Snyder (1989) Proc. Natl. Acad. Sci. USA 86: 9030-9033).
Endothelium derived NO relaxes the smooth muscles of blood vessels (Palmer et al. (1987) Nature 327: 524-526 and Ignarro et al. (1987) Proc. Natl. Acad. Sci. USA 84: 9265-9269) and inhibits platelet adhesion (Radomski et al. (1987) Biochem. Biophys. Res. Commun. 148: 1482-1489). NO production by cocultures of Kupffer cells and hepatocytes mediates inhibition of hepatocyte protein synthesis (Billiar et al. (1989) J. Exp. Med. 169: 1467-1472). NO is responsible for mediating the cytotoxic effects of macrophages and neutrophils (Hibbs et al. (1987) J. Immunol. 138: 550-556). NO has also been shown to be a major neuronal messenger in the brain (Bredt and Snyder (1989) Proc. Natl. Acad. Sci USA 86: 9030-9033). The meditation of functions of tissues as diverse as the brain, endothelium and blood cells indicates a wide-spread role for NO as a messenger molecule.
NO is formed by nitric oxide synthetase (NOS) from L-arginine with stoichiometric formation of L-citrulline. Studies have shown that a guanidino nitrogen of L-arginine is used to form NO (Iyengar et al. (1987) Proc. Natl. Acad. Sci USA 84: 6369-6373; Palmer et al. (1988) Nature 333: 664-666 and Marletta et al. (1988) Biochemistry 27: 8706-8711).
The formation of NO appears to involve the same or a similar enzyme in brain and endothelial cells but a different enzyme in macrophages. The brain-endothelium enzyme has been found to require calcium and calmodulin for activity (Bredt and Snyder (1990) Proc. Natl. Acad. Sci. USA 87: 682-685). The macrophage enzyme does not require calcium-calmodulin but does require tetrahydrobiopterin for activity (Tayeh and Marletta (1989) J. Biol. Chem. 264: 19654-19658; Soo Kwon et al. (1989) J. Biol. Chem. 264: 20496-20501).
The brain (i.e., calmodulin-dependent) NOS enzyme (EC 1.14.23.-) has been purified to homogeneity from rat brain, revealing a 150,000 kD protein (Bredt and Snyder (1990) Proc. Natl. Acad. Sci. USA 87: 682-685). The purification and molecular cloning of calmodulin-dependent NOS, as well as the preparation of antibodies immunoreactive with calmodulin-dependent NOS, is described in U.S. application Ser. No. 642,002, the disclosure of which is hereby incorporated by reference.
In addition to the differences between NOS activities in brain and endothelial cells as compared to macrophages, the regulation of NOS expression appears to differ as well. The synthesis of NO does not occur in macrophages unless they have been exposed to endotoxin (e.g., bacterial lipopolysaccharide) or cytokine (e.g., interferon-.gamma., -.beta. or -.alpha., tissue necrosis factor-.alpha. or -.beta.). However, in the brain and vascular endothelium, NOS is present without exposure to inducing agents (Knowles et al. (1990) Biochem, J. 270: 833-836). The arginine derivative L-N.sup..omega. -nitroarginine (NO.sub.2 Arg) has been described as being a competitive inhibitor of NOS (Moore (1990) Br. J. Pharmacol. 99: 408-412).
NO has been demonstrated to mediate neuronal relaxation of intestines (Bult et al. (1990) Nature 345: 346-347; Gillespie et al. (1989) Br. J. Pharmacol. 98: 1080-1082 and Ramagopal and Leighton (1989) Eur. J. Pharmacol. 174: 297-299) and to mediate stimulation by glutamate of cGMP formation (Bredt and Snyder (1989) Proc. Natl. Acad. Sci. USA 86: 9030-9033). Glutamate, the major excitatory neurotransmitter in the brain, acts through several receptor subtypes, some of which stimulate the formation of cGMP (Ferrendelli et al. (1974) J. Neurochem. 22: 535-540). Glutamate, acting at N-methyl-D-aspartate (NMDA) subtype of receptors, is responsible for neurotoxic damage in vascular strokes. Selective antagonists of NMDA glutamate receptors prevent neuronal cell death in animal models of hypoxic-ischemic brain injury (Choi (1990) J. Neurosci. 10: 2493-2501). Glutamate neurotoxicity has also been implicated in neurodegenerative disorders such as Alzheimer and Huntington diseases (Choi (1990) J. Neurosci. 10: 2493-2501 and Meldrum and Garthwaite (1990) Trends in Pharmacol. Sci. 11: 379-387).
An effective method of preventing, treating or ameliorating diseases caused by glutamate neurotoxicity is needed in the art.